Our proposal seeks to advance the field of distal gut microbiome research from its basis in genomics into quantitative proteomics through identification and characterization of bacterial proteins and enzymatic activities that are associated with inflammatory bowel diseases (IBD). The human intestinal tract harbors an enormous and diverse collection of commensal bacteria, termed the gut microbiome, that are essential for human metabolism, immune development and homeostasis, and epithelial cell angiogenesis. Recent studies demonstrate that aberrant levels of bacterially produced metabolites occur in IBD microbiota and are indicative of a pro-inflammatory predisposition. We hypothesize that the changes in metabolites are due to altered levels of bacterially secreted enzymatic activities arising from a dysfunctional IBD-associated distal gut microbiome. To test this hypothesis, we propose to design and use reactive chemical probes that irreversibly label enzymatic families and permit the isolation of these tagged proteins from complex proteomes. This enrichment technique is used in combination with mass spectrometry (MS) for protein identification and quantitation. As this is an initial proof-of-concept meta-proteomic study, we wil employ these chemical labeling techniques to an IL10-/- knockout IBD mouse model and analyze the murine distal gut microbiome. The IL10-/- mice microbiota will be probed for direct comparison to bacteria harvested from normal mice of an identical genetic lineage. To this end, we will exploit the use of SILAM (stable isotope labeling of mammals) to ensure quantitative measurement of identified proteins between healthy and IBD- associated commensal bacteria of these mouse models. Importantly, any differences in protein levels observed between healthy and IBD-associated mice populations may forecast deviations in proteins and enzyme functions in human IBD as metagenomic analyses have shown a high correlation of bacterial species and distribution within the microbiomes of human and mouse. Our innovative chemical biology and meta-proteomic methodologies are prerequisites to establish a foundation on which: 1) altered microbiomes of humans with IBD can be interrogated, 2) our understanding of microbiome homeostasis and alteration in intestinal inflammation can be improved, and 3) our ability to manipulate gut bacteria to combat a variety of human ailments can be enhanced. A successful outcome of our efforts will result in the first quantitative proteomic analysis of a microbiome-associated disease that can be adapted to humans, as well as result in a paradigm shift in approaches to chemical, biological, and biomedical investigations of the microbiome.